Using 10,000 Fragment Ions to Inform Scoring in Native Top-down Proteomics

Ashley N. Ives, Taojunfeng Su, Kenneth R. Durbin, Bryan P. Early, Henrique Dos Santos Seckler, Ryan T. Fellers, Richard D. Leduc, Luis F. Schachner, Steven M. Patrie, Neil L. Kelleher*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

18 Scopus citations

Abstract

Protein fragmentation is a critical component of top-down proteomics, enabling gene-specific protein identification and full proteoform characterization. The factors that influence protein fragmentation include precursor charge, structure, and primary sequence, which have been explored extensively for collision-induced dissociation (CID). Recently, noticeable differences in CID-based fragmentation were reported for native versus denatured proteins, motivating the need for scoring metrics that are tailored specifically to native top-down mass spectrometry (nTDMS). To this end, position and intensity were tracked for 10,252 fragment ions produced by higher-energy collisional dissociation (HCD) of 159 native monomers and 70 complexes. We used published structural data to explore the relationship between fragmentation and protein topology and revealed that fragmentation events occur at a large range of relative residue solvent accessibility. Additionally, our analysis found that fragment ions at sites with an N-terminal aspartic acid or a C-terminal proline make up on average 40 and 27%, respectively, of the total matched fragment ion intensity in nTDMS. Percent intensity contributed by each amino acid was determined and converted into weights to (1) update the previously published C-score and (2) construct a native Fragmentation Propensity Score. Both scoring systems showed an improvement in protein identification or characterization in comparison to traditional methods and overall increased confidence in results with fewer matched fragment ions but with high probability nTDMS fragmentation patterns. Given the rise of nTDMS as a tool for structural mass spectrometry, we forward these scoring metrics as new methods to enhance analysis of nTDMS data.

Original languageEnglish (US)
Pages (from-to)1398-1409
Number of pages12
JournalJournal of the American Society for Mass Spectrometry
Volume31
Issue number7
DOIs
StatePublished - Jul 1 2020

Funding

Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Awards T32GM105538 and R43GM130262 as well as the National Institute of Aging Award 1RF1AG063903-01. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

ASJC Scopus subject areas

  • Structural Biology
  • Spectroscopy

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